1. 1 CBER Assessment of Porcine Circovirus in Vaccines Philip R. Krause, M.D. FDA/CBER/OVRR 7 May 2010

2. 2 Introduction Historical Perspective –SV40 and polio vaccines –EAV in egg-derived vaccines –TSE issues and vaccines –Use of novel cell substrates for vaccines CBER Laboratory Assessment of Porcine Circovirus in vaccines

3. 3 SV40 and polio vaccines In 1960, Sweet & Hilleman discovered SV40 Formalin inactivation of IPV did not completely inactivate SV40 SV40-free vaccine was produced as quickly as possible –Licensure of OPV was delayed until SV40 free preparation could be made –No recall of IPV, but no release of additional IPV lots until SV40 was removed Consequences: –Some IPV recipients seroconverted to SV40 –OPV recipients (including those in clinical trials who were exposed to virus) did not seroconvert to SV40 –Concerns re: SV40 and human cancers– overall, evidence does not support a role

4. 4 EAV in egg-produced vaccines A more sensitive PCR test (PERT) for the enzyme, reverse transcriptase (RT), present in retroviruses was developed. In 1996, this test showed that previously undetectable quantities of RT were present in some avian cell- produced vaccines Additional studies showed that EAV (endogenous avian retrovirus) is a defective particle and does not induce productive infections in culture Long safety record of hens’ egg produced vaccines, together with absence of evidence of potential harm to humans were also important considerations

5. 5 TSE issues and vaccines In early 2000s, concern was raised that bovine derived materials sourced from countries with BSE or at risk of BSE could pose a theoretical risk to vaccinees Sourcing of bovine derived materials used for vaccine production was changed to countries not categorized as having BSE or at risk of BSE For products with potential exposures to bovine derived materials from countries with or at risk of BSE, risk assessments were performed, based on maximum possible exposures, dilution factors, potential infectious doses, etc. Many virus seeds were re-derived to remove all doubt

6. 6 Cell substrates Numerous discussions & advisory committee presentations on introduction of new cell substrates for vaccine production Discussions addressed issues posed by tumorigenic phenotype, theoretical infectivity and oncogenicity risks of cell DNA or other cell components Issues addressed by risk assessment

7. 7 Previous FDA evaluation of PCV CREON Pancreatic enzyme preparation –Advisory committee discussion 2008 –Discussion was broad-ranging, covering all potential porcine viruses– though PPV and PCV were recognized as the viruses most likely to survive the production process –Potential for exposure to porcine agents is mentioned in the package insert

8. 8 CBER’s Scientific Approach to Evaluating PCV in Vaccines Major question: Is PCV in rotavirus vaccines likely to represent infectious virus? –Molecular studies Can PCV nucleic acid be detected in vaccines? How much PCV nucleic acid is in vaccines? Are PCV nucleic acids in vaccines particle associated? Are PCV nucleic acids present as full-length genomes –In vaccine –In particles purified from vaccines How does sequence of PCV1 from vaccines compare with sequence of known infectious PCV1? –Cell culture studies Do vaccine-inoculated previously uninfected cells show evidence of virus infection?

9. 9 PCR assays used to detect PCV1 Four conventional PCR assays were used in initial studies We used sequencing & Southern hybridization to confirm results

10. 10 Rotarix contains PCV1 DNA Rotarix was consistently positive for PCV1 DNA Rotateq was negative for PCV1 DNA (to limits of this assay’s sensitivity) All assay controls (negatives, extraction controls, positives) yielded expected results These results are consistent with findings reported by GSK, Dr. Delwart

11. 11 Long PCR & Sequencing Long PCR –We detected and cloned a 1638 nt PCR product (PCV1 genome size: 1759 nt) directly from final Rotarix product Sequencing of PCV1 from Rotarix –Entire genome of virus from Rotarix final container available (combination of CBER and data from Dr. Delwart) –Some variability in sequence, but no nucleotide changes not already reported in Genbank for wild-type virus –No changes in ORFs PCV1 1638 nt

12. 12 Quantitative PCR Used three PCV1 primer pairs and two PCV2 primer pairs Studied DNA extracted from vaccine samples –Total DNA extracted from vaccines (“Direct prep”) –Particle-associated DNA was assessed by “Capsid prep”, which included nuclease treatment and ultracentrifugation Quantified PCV DNA by TaqMan qPCR PCV1 70 nt PCV1 529 nt PCV1 1638 nt PCV2 87 nt PCV2 357 nt

13. 13 PCV quantitation: Rotarix Both Rotarix samples were positive for PCV1 Both Rotarix samples were negative for PCV2 Not all virus DNA is full-length; longer PCRs yielded lower quantities of PCV1 DNA At each PCR fragment size, most PCV1 DNA was particle- associated Each vaccine dose contained around 100,000 copies of near full-length, particle-associated PCV1 DNA GSK 2GSK 3 PCV copies/mL 70 529 1638 357 87 ≤10 0 10 6 10 7 10 5 10 4 10 3 10 2 10 1 70 529 1638 357 87 LOQ Virus: PCV1 PCV2 Prep: Capsid Direct

14. 14 Further studies on Rotateq Using a more sensitive Taqman qPCR, PCV1 and PCV2 DNA fragments were detected in Rotateq final container Particle-associated PCV2 DNA fragments were detected in Rotateq bulks Further studies are ongoing

15. 15 GSK IPV-containing vaccines CBER’s final container PCRs for PCV1 and PCV2 are inconclusive due to PCR inhibition in spiking experiments –Follow up experiments pending Further studies will be performed when viral harvest samples are available –PCR –Infectivity

16. 16 Infectivity studies: Considerations Inactivation of rotavirus could also inactivate PCV Theoretical concern that bovine serum might contain neutralizing antibodies to PCV and thus reduce sensitivity of studies Need to assure that no exogenous PCV is added to culture Different cell types have different susceptibility to virus If cells become confluent, virus will not grow even in susceptible cell types Virus does not cause cytopathic effect S phase

17. 17 Infectivity studies: Considerations Inactivation of rotavirus could also inactivate PCV Theoretical concern that bovine serum might contain neutralizing antibodies to PCV and thus reduce sensitivity of studies Need to assure that no exogenous PCV is added to culture Different cell types have different susceptibility to virus If cells become confluent, virus will not grow even in susceptible cell types Virus does not cause cytopathic effect CBER approach Heat inactivation under conditions where PCV is resistant Use Ig-depleted bovine serum for cultures Use recombinant trypsin and pre- tested reagents Use swine testis (ST) cells Inoculate sub-confluent cells & maintain cells in growth phase by sub- passage @ day 3 Evaluate cultures by qPCR to detect virus DNA S phase

18. 18 Infectivity study: Rotarix Mock-infected Swine testis (ST) cells yielded negative results Cells inoculated with Rotarix (GSK1 and GSK 2) showed increasing PCV1 DNA quantities, measured by 529 nt qPCR Particle-associated PCV1 DNA was also produced (Not shown) Particle-associated PCV1 DNA was also detected in cell culture supernatants at day 3 and day 6 (Not shown) Inoculation of cell lysates from day 6 of vaccine cultures onto fresh ST cells revealed 2-4 log increases in PCV1 DNA quantity after 3 additional days in culture We have not formally evaluated the sensitivity of this assay ≤10 1 10 7 10 8 10 6 10 5 10 4 10 3 10 2 01234567 PCV1 copies Time (Days) Sample: GSK 2 GSK 3 Mock Prep: Capsid prep Direct extraction

19. 19 CBER and Manufacturer’s results VaccineCBERManufacturer Rotarix (GSK) PCV1 DNA in productPCV1 DNA in product, bulks, seeds, cells Particle-associated near full length PCV1 DNA in product Infectious PCV1 in cell culture IPV containing vaccines (GSK) PendingPCV1 DNA in harvest, seeds, cells, but not in bulks or final container Rotateq (Merck) Particle-associated PCV2 DNA in harvest; PCV1 and PCV2 DNA in final container PCV1 and PCV2 DNA in harvest, PCV DNA in final container

20. 20 Implications Tests of increasing sensitivity can detect very low quantities of virus DNA and of virus We do not know if it is possible to completely eliminate PCV from porcine trypsin Porcine and other animal-derived reagents used in vaccine manufacture could potentially contain other viruses, known and as-yet unknown It is impossible to absolutely assure that vaccines do not contain adventitious viruses

21. 21 Conclusions PCV1 DNA, particles, infectious virus are present in Rotarix PCV1 and PCV2 DNA is present at low levels in Rotateq –Further evaluation is ongoing PCV1 DNA is present in poliovirus harvests –Not detected by manufacturer in final bulks or final container –Quantities are low and purification/inactivation procedures are in place

22. 22 Laboratory work –Christine Uhlenhaut –Shasta McClenahan –Shuang Tang Reagents –Gordon Allan –Steve Krakowka –Andrew Cheung –XJ Meng FDA Scientific Advice –Jerry Weir –Robin Levis –Keith Peden –Arifa Khan –Konstantin Chumakov –Erik Henchal Acknowledgements

23. 23 DISCUSSION POINTS 1.Based on the information presented today on the detection of porcine circovirus (PCV) or PCV DNA in US licensed rotavirus vaccines: a)Please discuss the available scientific evidence and identify factors to be considered in assessing the potential risk of the use of US licensed rotavirus vaccines. b)Please discuss any additional scientific studies and information that you would recommend FDA consider in its deliberations.

24. 24 DISCUSSION POINTS 2. Given the available data about porcine circoviruses, including the lack of known infectivity and pathogenic effects in humans, and that porcine circoviruses or PCV DNA may be present in both US licensed rotavirus vaccines, please discuss factors to be considered in determining whether, or in what circumstances, the benefits of using rotavirus vaccines outweigh the theoretical risk from PCV.

25. 25 DISCUSSION POINTS 3.Please discuss the application of emerging technologies, and the implications for their use in the detection of known and unknown adventitious agents in vaccines currently licensed, as well as those under development.

26. 26 Backup slides

27. 27 PCV quantitation: Rotateq Weak signals were observed for both PCV1 and PCV2 DNA fragments in Rotateq final container This assay is more sensitive than that performed initially Virus DNA was not demonstrated to be particle-associated Near full-length PCV1 or 357 nt PCV2 sequences were not detected 70 529 1638 357 87 Merck 1Merck 2Merck 3 70 529 1638 357 8770 529 1638 357 87 PCV copies/mL ≤10 0 10 6 10 7 10 5 10 4 10 3 10 2 10 1 LOQ Virus: PCV1 PCV2 Prep: Capsid Direct

28. 28 PCV quantitation: Rotateq bulks Bulks are more concentrated than final container vaccines, so PCV concentration is expected to be higher In spiking experiments, inhibition was detected in the direct prep, but not in the capsid preps. Rotateq bulks were negative for particle-associated PCV1 Using the shortest (87 nt) PCR, some PCV2 DNA was particle-associated Bulk 1Bulk 2Bulk 3Bulk 4Bulk 5 PCV copies/mL 529 357 87 ≤10 0 10 6 10 7 10 5 10 4 10 3 10 2 10 1 529 357 87 529 357 87 529 357 87 529 357 87 LOQs Virus: PCV1 PCV2 Prep: Capsid Direct